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In Vitro Antibody Evolution by Ribosome Display

Ribosome display is powerful in vitro method for selection and evolution of antibodies from libraries since mutation can be continuously introduced into DNA pools in subsequent cycles. Creative Biolabs can provide antibody selection service and allow protein evolution during selection cycles. By ribosome display, it is possible to select and evolve high-affinity antibodies from very large libraries (up to 1014) with dissociation constants as low as 10-11 M in a very short time.

Overview of eukaryotic ribosome display selection and evolution of affinity Figure 1. Overview of eukaryotic ribosome display selection and evolution of affinity. (Conroy et al. 2009)

Antibody Evolution by Ribosome Display

In combination with DNA mutagenesis, ribosome display offers a powerful means of protein evolution in vitro. This approach offers a good way for antibody maturation and improving the biophysical properties. In vitro evolution has incomparable advantages over in vivo somatic maturation. It overcomes the limitation of nature and generates diversity of antibodies. Besides, in vitro evolution is a rapid and effective method for humanization of rodent antibodies, which allows simultaneous assessment of all mutated molecules.

In Vitro Evolution by Low-Fidelity DNA Polymerase

Ribosome display has enormous potential, which not only as a screening technique but also as an efficient method for real protein evolution. In this technique, in vitro evolution occurs by using non-proofreading polymerase in the step of PCR amplification and can be coupled with protein selection. By non-proofreading polymerase-based approach, selection round leads to a diversification of the DNA sequence pool. For instance, Taq DNA polymerase can be introduced to ribosome display process, which can lead to average one mutation per 20,000 nucleotides. Thus, through successive DNA diversification, followed by ribosome display/selection, we have the ability to produce antibody mutants with improved affinity and stability. The low-fidelity DNA polymerase-based method can selected antibody mutants with up to a 40-fold improvement in affinity over the original antibody fragment.

In Vitro Evolution by Other Methods

Except for using non-proofreading polymerase, diversification during PCR can be further enhanced by other methods, leading to the selection of evolved antibody fragments with improved properties.

Mutagenesis methods: oligonucleotide-directed mutagenesis or error-prone PCR in the presence of non-physiological metal ions such as Mn2+ or dNTP analogs.

Strategies for the introduction of mutations for improving affinity of recombinant antibodies Figure 2. Strategies for the introduction of mutations for improving affinity of recombinant antibodies (Conroy et al. 2009).

In vitro homologous recombination: such as DNA shuffling, and StEP (staggered extension process). The evolution of the sequence pool can be monitored by RIA. By recombination-based methods, deleterious or non-essential mutations can be suppressed by recombination with the original sequence, while beneficial mutations persist and accumulate. These methods can further be combined with random mutagenesis.

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  1. He M and Taussig M J (2002) “Ribosome display: cell-free protein display technology.” Brief Funct Genomic Proteomic 1(2):204-212.
  2. Schaffitzel C, Hanes J, Jermutus L, et al. (1999) “Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.” Immunol Methods 231(1-2):119-135.
  3. Conroy PJ, Hearty S, Leonard P et al. (2009) “Antibody production, design and use for biosensor-based applications.” Semin Cell Dev Biol 20(1):10-26. doi: 10.1016/j.semcdb.2009.01.010.

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